Figure 1.
Characterization of transgenic mice expressing OvPrP-A136 and OvPrP-V136. A.
Levels of transgene-expressed OvPrP in the CNS were estimated by semi-quantitative western blotting using mAb 6H4. Amounts of total protein loaded (µg) in each sample are shown. Prnp0/0, mice in which the PrP gene is disrupted; FVB, wild type mice. Estimates of expression levels, shown as a percentage (%) of that in FVB mice, are based on densitometric analysis of signals from diluted samples. B. Survival curves of mice following inoculation with sheep SSBP/1 scrapie prions. Percent (%) affected mice refers to numbers of mice within an inoculated cohort manifesting progressive clinical signs associated with prion disease. C. Western blot analysis of PK-treated brain extracts of diseased Tg(OvPrP-A136)3533+/− mice. SSBP/1 and CH1641 refer to mice inoculated with the respective prions. I and R refer to sheep SSBP/1 or CH1641 inocula, and brain extracts from recipient mice respectively.
Table 1.
Prion disease in transgenic mice expressing different ovine PrP scrapie susceptibility alleles.
Figure 2.
Analyses of PrPSc in the brains of SSBP/1 infected mice.
In A and C, densitometric analysis of immunoblots was used to measure the amounts of protease-resistant OvPrPSc as a function of GdnHCl concentration. The dose-response curve was plotted using a Gaussian non-linear least-square fit. Each point is the mean value derived from densitometric quantification of PK-resistant PrP in three diseased mouse brains. Error bars correspond to standard errors of the mean. A. Conformational stability analysis using mAb 6H4 of OvPrPSc-A136(S) in Tg(OvPrP-A136)3533+/− mice (red line), OvPrPSc-V136(U) in Tg(OvPrP-V136)4166+/− mice (blue line), and total OvPrPSc in Tg(OvPrP-A/V136) mice (black line). Black asterisks compare differences between OvPrPSc-A136(S) and total PrPSc in Tg(OvPrP-A136)3533+/− and Tg(OvPrP-A/V) mice; blue asterisks compare differences between OvPrPSc-A136(S) in Tg(OvPrP-A136)3533+/− mice and OvPrPSc-V136(U) in Tg(OvPrP-V136)4166+/− mice. C. Conformational stability analysis using mAb PRC5 of OvPrPSc-A136(S) in Tg(OvPrP-A136)3533+/− mice (solid line) and OvPrPSc-A136(U) in Tg(OvPrP-A/V136) mice (dashed line). *P<0.05, **P<0.005, ***P<0.001. In B and D, representative immunoblots of PK-resistant PrP in the brains of three mice from each infected cohort of Tg(OvPrP-A136)3533+/−, Tg(OvPrP-V136)4166+/−, and Tg(OvPrP-A/V136) mice using mAb 6H4 (B) and mAb PRC5 (C). Times of onset of disease for analyzed mice are also provided. OvPrP-V136 is indicated by blue symbols and text; OvPrP-A136 is indicated by red symbols and text.
Figure 3.
Representative OvPrPSc distribution in the CNS of diseased transgenic mice.
OvPrP-V136 and OvPrP-A136 are indicated by blue and red text respectively. Times of onset of disease (d) for individual mice analyzed in histoblots are provided. Sections through the midbrain, pons and oblongata are shown for SSBP/1 infected Tg(OvPrP-A136)3533+/− mice (A and D); Tg(OvPrP-V136)4166+/− mice (B and E); and Tg(OvPrP-A/V) mice (C and F). In panels G and H, sections were not treated with PK and show distribution of total PrP. Histoblots in panels A–G were probed with mAb 6H4; histoblots in D–H were probed with mAb PRC5.
Figure 4.
Detection of OvPrPSc by sPMCA.
In A and E, the first two lanes of each panel correspond to brain extracts from Tg(OvPrP-A136)3533+/− and Tg(OvPrP-V136)4166+/− mice respectively without PK treatment. A136, V136, and A136/V136 refer to substrates from Tg(OvPrP-A136)3533+/− mice, Tg(OvPrP-V136)4166+/− mice, and mixtures of the two respectively. In B and F, the first lanes of each panel correspond to brain extracts from Tg(OvPrP-A136)3533+/− mice without PK treatment; in C and G, the first lanes of each panel correspond to brain extracts from Tg(OvPrP-V136)4166+/− mice without PK treatment; in D and H, the first lanes of each panel correspond to mixtures of brain extracts from Tg(OvPrP-A136)3533+/− and Tg(OvPrP-V136)4166+/− without PK treatment. In B–D and F–H, three different brains, indicated by #1–#3 were used as sources of substrates. In each case, the second lane of the second panel contains PK treated SSBP/1. Panels A and E are sPMCA using the indicated templates in the absence of a prion seed. Numbers in black above each lane refer to the round of sPMCA. C refers to a control in which no SSBP/1 seed was added. In each case C, and all numbered lanes were treated with PK. Blots were probed with mAbs 6H4 or PRC5 as indicated.
Figure 5.
PMCA using defined seeds and substrates.
PMCA was performed for various times indicated. At each time point samples were either amplified by sonication (A), or matching control samples (C) received no sonication, and were therefore not amplified. Brain homogenates from healthy Tg(OvPrP-A136)3533+/− and Tg(OvPrP-V136)4166+/− mice served as sources of OvPrP-A136, OvPrP-V136 or mixtures of the two. In A, samples were seeded with sheep SSBP/1; in B, samples were seeded with brain extracts from diseased Tg(OvPrP-V136)4166+/− mice [SSBP/1-V136(U) prions]; in C, samples were seeded with brain extracts from diseased Tg(OvPrP-A136)3533+/− mice [SSBP/1-A136(S); in D, samples were seeded with extracts from diseased Tg(OvPrP-A/V) mice. Samples labeled 12* received no seed. The first three lanes of each immunoblot were loaded with the substrate(s) for each PMCA reaction, not treated with PK; the corresponding seed, not treated with PK; and, the same seed treated with PK. All other samples were digested with PK. Western blots were probed with mAbs 6H4 and PRC5 as indicated.